(1) Field of the Invention
The present invention relates to the formation of fluorescent linkages, and more particularly to the formation of chemoselective fluorescent linkages between compounds, including conjugation of biomolecules.
(2) Description of the Related Art
It is often useful to be able to measure the degree of attachment of certain compounds to certain other compounds. This selective binding, or conjugation, is particularly useful in biological assays and diagnostic procedures in which marker compounds selectively bind to target compounds. If the marker compound has properties that can be measured by some sensing method, then its presence can be measured, thereby providing an indirect measurement of the target compound to which it is bound. This technique has been of significant utility in research in the life sciences in methods such as in vivo imaging, drug delivery, biochemical assays, bioengineering, cell-based assay and microarrays, and the like. For general information, see, for example, Saxon, E. et al., Science, 287: 2007-10 (2000), and Breinbauer, R. et al., Chembiochem 4:1147-9 (2003).
Bioconjugation technology affects almost every discipline of the life sciences, such as drug discovery, proteomics and cell biology. Bioconjugation aims at the linking of two or more molecules (or supramolecules) to form a new complex with the combined properties of its individual components. See Hermanson, G. T. et al., Bioconjugate Techniques, Academic Press, San Diego (1996). One important application of bioconjugation is to modify cellular components selectively with signaling probes for proteomics, functional genetics, and cell biology research.
A multistep procedure is commonly employed wherein the cellular entity is first attached with a detectable tag, such as fluorescent dyes or biotin, followed by purification of the ligated product and detection. However, excess prelabled reagents are generally hard to remove from the intracellular environment or from tissues of living organisms, which prohibits the application of a multistep labeling procedure in many situations.
While several bioconjugation techniques are widely used, most of them rely on the reaction between electrophiles and nucleophiles, which can hardly offer truly chemoselective reactions. In addition, the lack of an efficient way to detect and evaluate bioconjugation efficiencies is the big obstacle of many applications. The common way for applying this method of detection is a two-step or three-step procedure. First, the conjugation will introduce the primary labeling reagents, such as biotin and peptide tags. After the purification, the fluorescent secondary reagents will be used to give the detectable signals.
An ideal alternative to present bioconjugation systems would be a chemoselective process that was unaffected by biological components, and which used reagents which, while unbound, did not contribute a significant background signal, but which resulted in a ligated product that provided a strong detectable signal. Very few such systems have been reported.
One system, reported by Gaietta et al., Science, 296:503-507 (2002), utilized the synthesis and application of a biarsenical fluorophore which can bind with a short peptide sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- with high affinity, then become strongly fluorescent. Even though the in vivo test provided good results, the method suffers from the high concentration of biarsenical compounds needed to overcome the non-specific finding from other cysteine residues as well as the high toxicity of the biarsenical reagents.
In other work, Lemieux et al., J. Am. Chem. Soc., 125:4708-4709 (2003), or Saxon et al., Science, 287:2007-2010 (2000), report the use of a fluorogenic coumarin-phosphine that is activated by the Staudinger reaction with azides. This yielded a coumarin-phosphine product compound that was unstable to oxidation that can give rise to a high level of false background signals. Also, phosphine is not a bio-friendly reagent. Therefore, such a system would appear to have limited applications in real biomedical situations.
Recent advances in the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes has provided promising reactions that appear to afford regioselectivity and almost quantitative transformation under mild conditions. See, e.g., Rostovtsev et al., Angew. Chem., Int. Ed., 41:2596-2599 (2002), or Tornoe et al., J. Org. Chem., 67:3057-3062 (2002). Several groups have used these compounds as a pair of linkers for chemoselective ligations through a Cu(I)-mediated cycloaddition reaction. See, e.g., Wang et al., J. Am. Chem. Soc, 125:3192-3193 (2003), or Speers et al., J. Am. Chem. Soc., 125:4684-4687 (2003), or Seo et al., J. Org. Chem., 68:609-612 (2003), or Lee et al., J. Am. Chem. Soc., 125:9588-9589 (2003), or Link, et al., J. Am. Chem. Soc., 125:11164-11165 (2003), or Deiters et al., J. Am. Chem. Soc., 125:11782-11783 (2003), or Brienbauer et al., ChemBioChem, 4:1147-1149 (2003).
However, the need remains for a bioconjugation method that would have a high chemoselectivity, but which did not affect and was unaffected by biological components, and which used reagents which, while unbound, did not contribute a significant background signal, but which resulted in a ligated product that provided a strong detectable signal.